Allogeneic, donor-derived, second-generation, CD19-directed CAR-T cells for the treatment of pediatric relapsed/refractory BCP-ALL.

Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) or displaying profound lymphopenia and/or rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells.

Intrahepatic Administration of Human Liver Stem Cells in Infants with Inherited Neonatal-Onset Hyperammonemia: A Phase I Study.

Previous studies have shown that human liver stem-like cells (HLSCs) may undergo differentiation in vitro into urea producing hepatocytes and in vivo may sustain liver function in models of experimentally induced acute liver injury. The aim of this study was to assess the safety of HLSCs intrahepatic administration in inherited neonatal-onset hyperammonemia. The study was approved by the Agenzia Italiana del Farmaco on favorable opinion of the Italian Institute of Health as an open-label, prospective, uncontrolled, monocentric Phase I study (HLSC 01-11, EudraCT-No.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

Background Aims: Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS).

Validation of analytical methods in compliance with good manufacturing practice: a practical approach.

Background: The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range.

Methods: For the endotoxin test we used a kinetic chromogenic LAL test.

Myogenic potential of whole bone marrow mesenchymal stem cells in vitro and in vivo for usage in urinary incontinence.

Urinary incontinence, defined as the complaint of any involuntary loss of urine, is a pathological condition, which affects 30% females and 15% males over 60, often following a progressive decrease of rhabdosphincter cells due to increasing age or secondary to damage to the pelvic floor musculature, connective tissue and/or nerves. Recently, stem cell therapy has been proposed as a source for cell replacement and for trophic support to the sphincter. To develop new therapeutic strategies for urinary incontinence, we studied the interaction between mesenchymal stem cells (MSCs) and muscle cells in vitro; thereafter, aiming at a clinical usage, we analyzed the supporting role of MSCs for muscle cells in vitro and in in vivo xenotransplantation.

Effect of In Vitro Exposure of Corticosteroid Drugs, Conventionally Used in AMD Treatment, on Mesenchymal Stem Cells.

Age-related macular degeneration (AMD) is a leading cause of legal blindness in individuals over 60 years of age, characterized by the dysfunction of retinal pigmented epithelium cells, specifically in the macular area. Despite several treatment options, AMD therapy remains difficult, especially for exudative AMD. Multipotent mesenchymal stem cells (MSCs), with great plasticity and immunomodulant properties, are a promising cell source for cellular therapy and tissue engineering.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting.

Background: The quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests' accuracy, precision, repeatability, linearity and range.

Methods: As the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2.

Intrabone cord blood hematopoietic stem cell transplantation in a subset of very high-risk pediatric patients: a safety and feasibility pilot study.

The main limit of umbilical cord blood hematopoietic stem cell transplantation is a more difficult engraftment related to the number of cells infused per kilogram of recipient body weight. This limit makes the cord blood a suboptimal source of hematopoietic stem cells for transplantation in case of difficult engraftment situations. Direct intrabone cord blood (CB) injection has been recently investigated as a solution to cell dose problem in the adults population, but there is a lack of data concerning this approach in pediatric patients.

Multipotent mesenchymal stromal stem cell expansion by plating whole bone marrow at a low cellular density: a more advantageous method for clinical use.

Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of "optimal" conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods.

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies.

Background Aims: Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.

Bone marrow mesenchymal stem cells from healthy donors and sporadic amyotrophic lateral sclerosis patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective therapies. Cell replacement therapy has been suggested as a promising therapeutic approach for multiple neurodegenerative diseases, including motor neuron disease. We analyzed expanded mesenchymal stem cells (MSCs) isolated from sporadic ALS patients and compared them with MSCs isolated from healthy donors.

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors.

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced.

Refreezing of cord blood hematopoietic stem cells for allogenic transplantation: in vitro and in vivo validation of a clinical phase I/II protocol in European and Italian Good Manufacturing Practice conditions.

Objective: Several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (HSCs). Because most cord blood (CB) samples are frozen in single bags and only an aliquot ( approximately 25%) of the blood can be expanded, the thawing and refreezing of samples must be validated in the current European and Italian Good Manufacturing Practice (eIGMP) conditions. Here, we describe in vitro and in vivo validation of the phase I/II protocol for CD34+ expansion of thawed CB units according to the current Cell Therapy Products (CTPs) Guidelines.

Serial transplantations in nonobese diabetic/severe combined immunodeficiency mice of transduced human CD34+ cord blood cells: efficient oncoretroviral gene transfer and ex vivo expansion under serum-free conditions.

Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated.

Fast but durable megakaryocyte repopulation and platelet production in NOD/SCID mice transplanted with ex-vivo expanded human cord blood CD34+ cells.

We have previously established a stroma-free culture with Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) that allows the maintenance and the expansion for several weeks of a cord blood (CB) CD34+ cell population capable of multilineage and long-lasting hematopoietic repopulation in non-obese diabetic/ severe combined immunodeficient (NOD/SCID) mice. In this work the kinetics of megakarocyte (Mk)-engraftment that is often poor and delayed in CB transplantation, and human platelet (HuPlt) generation in NOD/SCID mice of baseline CD34+ cells (b34+), and of CD34+ cells reisolated after a 4-week expansion with FL+SCF+TPO (4w34+) were compared. With b34+ cells Mk-engraftment was first seen at week 3 (CD41+: 0.

Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication.

Telomerase activity, telomere length, stem/progenitor cell production, and function of CD34+ cells from cord blood (CB), bone marrow, and mobilized peripheral blood were evaluated in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34+ cells reisolated at monthly intervals for passage. Continuous expansion of stem cells as measured by in vitro cobblestone area and secondary colony-forming assays was noted for 18 to 20 weeks and by severe combined immunodeficiency (SCID)-repopulating cells (SRCs), capable of repopulating and serially passage in nonobese diabetic/SCID mice, for 16 weeks.

In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells: rapid and transient megakaryocyte reconstitution.

Background And Objectives: Megakaryocyte (Mk) engraftment is often poor and delayed after cord blood (CB) transplantation. Ex vivo manipulations of the cells that will be infused may be a way to achieve better Mk engraftment. In this study we investigated the ability of different hematopoietic growth factor combinations to generate large numbers of Mk cells ex vivo.

Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution.

Objective: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described.

Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient.

The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells.

Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation.

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34(+) stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34(+)CD38(-)CXCR4(-) and CXCR4(+) cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34(+)CXCR4(+) (R4(+)) and CD34(+)CXCR4(-) (R4(-)) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34(+) cells.